In cardiac excitation-contraction coupling, the sarcoplasmic reticulum (SR) plays an essential role in the regulation of the cytosolic free Ca2+ concentration. There are three major functions of the SR: a) Ca2+-uptake from the cytosol into the SR lumen resulting in muscle relaxation; b) Ca2+ storage in the SR lumen; and c) Ca2+-release from the SR into the cytosol resulting in muscle contraction. The main SR proteins responsible for these functions are: the Ca2+-transport ATPase (SERCA), the Ca2+ storage protein calsequestrin, and the Ca2+ release channel or ryanodine receptor, respectively. Phospholamban is another SR protein, which plays a crucial role in the regulation of the Ca2+-ATPase activity and myocardial contractility. In this project, we propose further studies on elucidating the regulatory role of PLB in the mammalian heart and defining the stoichiometric coupling ratio between PLB and the Ca2+-pump, which appears to be a key determinant of cardiac contractile parameters. We also propose to elucidate the role of the PLB phosphorylation status, through regulation of its phosphatase activity by inhibitor 1, in the control of contractility under basal and beta-agonist conditions. Furthermore, since alterations in the levels of PLB or in the degree of PLB phosphorylation reflect alterations in SR Ca2+ load and contractility, we propose to elucidate the functional role of SR Ca2+ load through calsequestrin, the major Ca2+ storage protein in the SR lumen. Animal models with alterations in the expression levels of this protein (overexpression and knockouts) will be generated and their cardiac phenotype will be analyzed at the subcellular, cellular, organ and intact animal levels. These studies will provide important information on the physiological role of calsequestrin in vivo. Overall, our proposed studies will advance our knowledge on the mechanisms underlying regulation of Ca2+ homeostasis by the SR function. They will also provide valuable insights into the crosstalk between the various SR Ca2+ handling proteins and their regulatory effects on cardiac contractility.